Stem cells have enormous potential in tissue engineering and regenerative medicine. However, in order to use stem cells to generate tissue for implantation, we must overcome a major challenge, which is to provide a functional vascular network and adequate perfusion while the tissue is growing. To achieve this long term goal of generating functional vasculature from stem cells, our first step is studying how to control stem cell differentiation into the proper vascular lineages. During normal vascular development, embryonic stem cell commitment into vascular lineage and further into the arterial and venous cell fate is crucial to define the patterns of vascular network and facilitate the perfusion when the heart starts to beat. Blood flow is another important driving force to maintain vascular identity and stimulate vascular maturation into a functional state. Therefore, to engineer a functional vasculature from stem cells, it is important to recapitulate these critical steps in vascular development process. In this proposal, we plan to investigate the hypothesis that simultaneously differentiating stem cells into arterial and venous endothelial cells and further maintaining their identity are critical for building a functional vascular network. The goal of this study is to build a model system that allows us to test this hypothesis and to define critical factors for controlling the spatial patterns of arterial and venous cell fate. In Specific Aim 1, we will develop a 3-Dimensional cell printing technology capable of providing on-demand control of stem cell niches. In Specific Aim 2, we will use this technology to engineer an in vitro environment that recapitulates many signal cues important for normal vascular development, and study their effects on embryonic stem cell differentiation into the arterial and venous specification. Based on these studies, we expect to define critical factors that differentially control arterial and venous cell fate. In Specific Aim 3, we will incorporate these signals to build a 3-Dimensional Arterial-Venous loop to support the growth of perfused microvascular network derived from embryonic stem cells. Our research undertakes a first step toward the goal of engineering a functional vasculature by focusing on how to differentiate embryonic stem cells into the proper vascular lineage using a novel cell printing technology. In addition, our study will create an in vitro model of human vascular development which can reveal some detailed information of early developmental events and may provide novel insights into the mechanisms of various forms of vascular anomalies. PUBLIC HEALTH RELEVANCE: This project will use a novel cell printing technology to differentiate embryonic stem cells into arterial and venous cells, and further assemble them into 3-D vascular network with perfusion, which is a critical step in engineering human tissues for therapeutic purpose. In addition, this research will also reveal information related to early vascular development process and may provide insight into the mechanisms of arterial-venous malformations.